Synthetic Lethality Effects Of Ikk? Inhibition And Base Excision Repair Blockage On AML Cells

Purpose : To investigate the synthetic lethality effects of Ikk? inhibition and base excision repair(BER)blockage on damaged AML cells and its mechanism.Methods:(1)MTT assay was used to determine the sensitivity of AML cells to BMS-345541 and Olaparib,MTT assay was also used to detect the synthetic lethality effects of two drugs on DNA damaged AML cells.(2)Apotosis was analyzed with FITC-Annexin V Apotosis Detection Kit by Flow Cytometry(FCM).(3)The level of DNA damage in AML cells was tested by FCM.(4)Establish a stable knockout HL-60PARP-1(-)cell line by small hairpin RNA(sh RNA)targeting PARP-1.MTT assay and flow cytometry were applied to detect the difference of DNA damage repair between HL-60 Vector and HL-60PARP-1(-)cells.(5)High Content Analysis(HCA)was used to verified the amount of ?-H2 AX in AML cells.(6)The HR assay was carried out using the p DR-GFP reporter assay.(7)The m RNA level of BRCA-1 and PARP-1was examined by Real-time PCR.(8)Western blot was used to detect p-ATM level.(9)HCA was used to detect the level of PAR in AML cells.(10)Establish an HL-60 cell model of subcutaneous xenotransplanted tumor to examine whether there is synthetic lethality effects to DNA damaged AML cells in vivo.Res?Lts:(1)BMS-345541 and Olaparib can significantly inhibit the proliferation of AML cells treated with DNR and do have synthetic lethality effects between two drugs.CI values<1.(2)BMS-345541 and Olaparib increased the apoptosis rate in AML cells treated with DNR,while the synthetic lethality group increased much more.(3)FCM analysis showed that BMS-345541 and Olaparib increased the amount of ?-H2 AX foci in AML cells treated with DNR,while the synthetic lethality group increased much more.(4)The results of MTT and FCM showed that PARP-1knockdown inhibited DNA damage repair in HL-60 cells,increased its apoptosis rate.(5)HCA analysis showed that BMS-345541 and Olaparib increased the amount of?-H2 AX foci and p-ATM foci in AML cells treated with DNR after 24 h,while the synthetic lethality group increased much more.(6)The HR assay showed that BMS-345541 blocked HR repair in damaged AML cells.(7)The expression of BRCA-1 in was decreased after treated with BMS-345541 tested by Real-Time PCR.(8)DNA damage related protein p-ATM level raised in BMS-345541 treated group.(9)HCA analysis showed that Olaparib decreased the amount of PAR in AML cells.(10)BMS-345541 and Olaparib can significantly inhibit the proliferation of subcutaneous xenotransplanted tumor model of HL-60 cell,and prolong the survival length of mice.Conclusion: DNR leads to DNA damage in AML cells.Olaparib can inhibit single-strand breaks repair of damaged DNA by blocking BER pathway,and BMS-345541 can inhibit double-strand breaks repair of damaged DNA by inhibiting HR pathway.Ikk? inhibition and BER Blockage enhance the apoptosis induced by DNR,and do have synthetic lethal effect.